The objective of this proposed research is to develop and utilize novel microfluidic devices in concert with established imaging methods in order to elucidate intercellular signaling dynamics among intact pancreatic islets of Langerhans. Little is known about possible mechanisms that coordinate oscillations throughout multiple islets in the pancreas, but the globally pulsatile secretion that has been observed in the pancreas of whole animals and humans suggests an obvious coordination among these islets. Since this pulsatile behavior is disrupted in diabetic patients, the elucidation of these mechanisms could provide considerable insight into the diabetic condition. In this proposal, it is hypothesized that paracrine signaling plays a major role in coordinating oscillatory behavior among multiple islets. With the typical islet diameter in the range of 100-200 micrometers, the channel dimensions in most commonly used microfluidic devices prove to be ideal for isolating and studying these particular cells. Thus, microfluidic tools will be utilized to investigate this coordination by creating inter-islet communication networks that can be switched on or off using on-chip valves. In Specific Aim 1, microfluidic architectures, flow control methods, and cell manipulation strategies will be optimized to correlate zinc ion concentration with insulin secretion from single live islets. Non- pulsatile flow control methods previously developed by the applicant will be used to stimulate islets bathed in zinc-indicator dye to allow quantitative, real-time imaging of insulin secretion from individual islets. In Specific Aim 2, the role of paracrine signaling in intercellular oscillatory behavior among multiple live islets will be determined. The novel microfluidic tools developed in Specific Aim 1 will be used to position multiple islets into communication networks with precisely controlled, non-pulsatile stimulation. These experiments will compare communication among normal islets, pharmacologically altered normal islets, and islets from genetically manipulated (3IRKO mice, which have lost insulin secretory activity, resembling islets from mice with Type II diabetes. This project is specially designed to provide training in quantitative imaging and biological methods through use of the applicant's expertise in micro-analytical methodology. The importance of understanding these types of mechanistic pathways of islet behavior can not be overstated. Diabetes affects 20.8 million children and adults in the United States (7.0% of population), and cost an estimated $132 billion in 2002; the disorder can result in serious complications such as heart disease, stroke, blindness, kidney disease, and nervous system disease. [unreadable] [unreadable] [unreadable]